For scientific, regulatory, or other applications, many persons, institutions, and agencies often require analyses of samples to determine whether such small molecule and ionic analytes are present. Examples of such analyses include the determination of metal ions in sea water to understand the processes of chemical oceanography; determination of toxic materials such as Hg(II), Ni(II), CN.sup.-, or HS.sup.- in groundwater or wastewater; detection of the corrosion of metal alloys by the presence of Co(II), Zn(II), or Cu(II) in condensates; or the presence of metals in lubricating oil as an indicator of machinery wear and incipient failure.
Many methods are known in the art for such analyses. For metal ions, such methods include graphite furnace atomic absorption spectrophotometry, inductively coupled plasma atomic emission spectroscopy and mass spectroscopy, various electrochemical means, and photoluminescence spectroscopy using metallo-photoluminescent indicators. For common anionic analytes there are fewer techniques available; they include ion chromatography, mass spectrometry, and electrochemical means.
Most of these techniques involve analysis of single or multiple discrete samples in a specialized instrument which may not be close to the sample. This is a particular drawback for analytical tasks that require a continuous or quasi-continuous determination of the analyte with real time readout of the result; require samples to be collected from remote, inaccessible, or hazardous environments; or require such extensive sampling that it is prohibitively costly. For many of these methods a sensor capable of remotely, continuously, and selectively monitoring the analyte of interest in situ is required, and subsequent reporting of the results of the analysis back to the operator in real time.
Improvements have been made in the development of photoluminescence-based sensors for a variety of applications (Thompson, R. B. (1991) in Topics in Fluorescence Spectroscopy, Vol. 2: Principles, Lakowicz, J. R. (Ed.) Plenum Press, NY; Wolfbeis, O. S. (Ed.) (1992) Fiber Optic Chemical Sensors and Biosensors Vols. I and II, CRC Press, Boca Raton, Fla.; Lakowicz, J. R., and Thompson, R. B. (Eds.) (1993) Proc. of the SPIE Conference on Advances in Fluorescence Sensing Technology Vol. 1885, Society of Photooptical Instrumentation Engineers, Bellingham, Wash.) A central issue in the development of such sensors has been the means of transduction, whereby the presence or relative amount of the chemical analyte is transduced as a change in the photoluminescence which may be quantitated. Thus, workers in the field have mainly transduced analyte levels as changes in photoluminescence intensity (Thompson (1991); Saari, L. A., and Seitz, W. R. (1982) Anal. Chem. 54, 821; Thompson, R. B. and Ligler, F. S. (1991) in Biosensors with Fiber Optics., Wise, D., and Wingard, L. (Eds.) pp. 111-138, Humana Press, Clifton, N.J.), or ratios of photoluminescence intensity at two different wavelengths (wavelength ratiometric) (Tsien, R. Y. (1989) Ann. Rev. Neurosci. 12, 227; Opitz, N., and Lubbers, D. W. (1984) Adv. Exp. Med. Biol. 180, 757; Thompson, R. B. and Jones, E. R. (1993) Anal. Chem. 65, 730-4; and U.S. Pat. No. 5,545,517).
The ratio approach has proven particularly popular because it is robust in avoiding many of the artifacts which limit the accuracy and precision of simple intensity measurements. The major limitation of the ratio approach has been the limited number of ratiometric photoluminescent indicators (Haugland, R. P. (1992) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, Oreg.).
Intensity based methods, however, are sensitive to artifacts, as any change in photoluminescence intensity, regardless of its origin may be misinterpreted as a change in concentration. Changes in light scattering, or variations in excitation light intensity and/or photobleaching may easily be misinterpreted as a change in the concentration of the metal ion. Although the accuracy and precision of these methods may be improved through the use of internal photoluminescent standards, or with the monitoring of excitation intensity or with the use of kinetic methods of analysis, careful and even repeated calibration of reagents and instrumentation are required to minimize spurious variations in signal intensity.
Recently, many groups have shown that transducing the level of analyte as a change in photoluminescence lifetime is a robust and flexible approach to optical sensing (Demas, J. N., (1994) in Topics in Fluorescence Spectroscopy, Vol. 4: Probe Design and Chemical Sensing, Lakowicz, J. R. (Ed.) Plenum Press, NY; Lippitsch, M. E., Pusterhofer, J., Leiner, M. J. P. and Wolfbeis, O. S. (1988) Anal. Chim. Acta 205, 1-6; Keating, S. M., and Wensel, T. G. (1991) Biophys. J. 59, 186-202; Lakowicz, J. R., Szmacinski, H., and Karakelle, M. (1993) Anal. Chim. Acta 272, 179-186; Szmacinski, H., and Lakowicz, J. R. (1993) Anal. Chem. 65, 1668-74; Lakowicz, J. R. (1992) Laser Focus World 28(5) 60-80; Thompson, R. B. and Patchan, M. W. (1993) in Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V. Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.; Ozinskas, A. J., Malak, H., Joshi, J., Szmacinski, H., Britz, J., Thompson, R. B., Koen, P. A., and Lakowicz, J. R. (1993) Anal. Biochem. 213, 264-270). Lifetime-based sensing can exhibit a dynamic range of greater than five orders of magnitude in analyte concentration (Szmacinski, H., and Lakowicz, J. R. (1993) Anal. Chem. 65, 1668-74; Thompson, R. B. and Patchan, M. W. (1993) in Proc. of the SPIE Conference on Chemical, Biochemical, and Environmental Fiber Optic Sensors V. Lieberman, R. A. (Ed.) pp. 296-306. Society of Photooptical Instrumentation Engineers, Bellingham, Wash.). Lifetime-based sensing has been adapted to and has particular advantages for fiber optic sensors.
Photoluminescence resonance energy transfer is a dipole-dipole interaction described by Forster (Forster, Th. (1948) Ann. Physik 2, 55-75) that is very useful for lifetime-based sensing. Forster's theory is very well-established, with thousands of examples in the literature of its predictive power. The rate of energy transfer K.sub.T is a function of the distance between donor and acceptor r, the refractive index of the medium n, the degree of energy overlap J between the emission spectrum of the donor and the absorbance spectrum of the acceptor, the emissive rate of the donor in the absence of acceptor I.sub.d, and the relative orientation between the donor and acceptor dipoles k.sup.2 : EQU K.sub.T =(r.sup.-6 Jk.sup.2 n.sup.-4 l.sub.d).times.8.71.times.10.sup.23 sec.sup.-1
The rate of energy transfer can be simply expressed in terms of a Forster distance R.sub.0, which is the distance at which the rates of emission and energy transfer are equal, and the lifetime of the donor .tau. (tau): ##EQU1##
Although, lifetime and ratiometric methods are qualitatively similar with respect to their freedom from spurious variations in photoluminescence intensity, the physical measurement of photoluminescence lifetime is costlier and technically more difficult than a simple intensity measurement, especially in the context of imaging applications. Regardless of these drawbacks, lifetime analysis has been preferred for reasons that include its broad dynamic test range in sample concentrations which may exceed five orders of magnitude in some cases. Lifetime-based analysis also minimizes errors associated with optics, fluorophore concentration and detector sensitivity.
In the '351 patent application identified above, we have introduced the use of photoluminescence polarization (anisotropy) for the detection and quantitative analysis of metal ions in aqueous solutions. It is simply predicated on the use of a photoluminescent indicator system that will emit polarized light in a measurably different manner on the formation of a macromolecule-metal ion complex. This complex may rely on either the metal dependent binding of a photoluminescent indicator to a macromolecule, or the binding of a metal ion to a macromolecule that has previously been labeled with a photoluminescent label. Thus, using the teachings described in our previous patent application, one can detect and quantitate metal ions in solution by a homogeneous photoluminescence polarization (anisotropy) assay based on proximity dependent quenching mechanisms and other means, and not simply to a change in the rotational correlation time of the fluorescent label.
A labeled macromolecule such as carbonic anhydrase, wherein the donor fluorophore is approximately at its Forster distance from a suitable metal in the active site, would exhibit a more rapid apparent decay of its photoluminescence due to energy transfer; energy transfer is thus a quenching mechanism, and a decrease in photoluminescence intensity should be observed as well. It is well known that by placing the donor much closer to the active site, essentially quantitative quenching results, whereas if the donor is much further away, the quenching is modest due to the sixth power dependence. Additionally, we have identified certain embodiments that exhibit metal-dependent fluorescence quenching which cannot be due to Forster transfer and which therefore quench by other proximity-dependent mechanisms. These embodiments, described in greater detail below, include but are not limited to photoluminescent sensors for lifetime sensing of cadmium and zinc comprised of the N67C variant of carbonic anhydrase variant labeled with ABD-T or ABD-F.
In the '904 application mentioned above, we first demonstrated that positioning of the photoluminescent donor moiety on the macromolecule at particular distances from a metal ion bound to the macromolecule or an inhibitor bound to the metal ion optimizes the response of the assay. Thus, a sensor, which we term "photoluminescence-based biosensor", can transduce the presence or level of the metal ion or ligand as a change in the photoluminescence of an indicator phase, which can be measured through a length of optical fiber with the indicator phase at the distal end in contact with a sample containing the metal ion or ligand at the proximal end. Our previous applications demonstrated that the determination of metal ions and ligands such as anions using a photoluminescent energy transfer mechanism employing a macromolecule. We also demonstrated the importance of controlling the proximity of the photoluminescent-donor-label to the metal ion or ligand in making the determinations.
The invention herein, described in detail below, is directed to new and improved photoluminescence-based biosensors for use in the photoluminescence detection methods described in co-pending patent applications recited above. These detection methods include change in intensity, wavelength, polarization, and lifetime (time-dependance) of the emission.